Regulatory control of gene expression has received special attention by scientists. In special circumstances, gene expression has been achieved by employing recombinant DNA as well as other techniques.
For example, in the PCT Patent Application WO 83/01451, published Apr. 23, 1983, there is disclosed a technique employing an oligonucleotide, preferably in phosphotriester form having a base sequence substantially complementary to a portion of messenger ribonucleic acid (mRNA) coding for a biological component of an organism. This oligonucleotide is introduced into the organism and, due to the complementary relationship between the oligonucleotide and the messenger ribonucleotide, the two components hybridize under appropriate conditions to control or inhibit synthesis of the organism's biological component coded for by the messenger ribonucleotide. If the biological component is vital to the organism's viability, then the oligonucleotide could act as an antibiotic. A related technique for the regulation of gene expression in an organism is described in Simons, et al., "Translational Control of IS10 Transposition", Cell 34, 683-691 (1983). The disclosures of the above-identified publications are herein incorporated and made part of this disclosure.
In U.S. patent application Ser. No. 543,528 filed Oct. 20, 1983 of which this application is in turn a continuation-in-part, gene expression is regulated, inhibited and/or controlled by incorporating in or along with the genetic material of the organism, DNA which is transcribed to produce an mRNA having at least a portion complementary to or capable of hybridizing with an mRNA of said organism, such that upon binding or hybridizing to the mRNA, the translation of the mRNA is inhibited and/or prevented. Consequently, production of the protein coded for by the mRNA is precluded. In the instance here, because the mRNA codes for a protein vital to the growth of the organism, the organism becomes disabled. It is also disclosed that this technique for regulating or inhibiting gene expression is applicable to both prokaryotic and eukaryotic organisms, including yeast.
As indicated hereinabove, it is known that the expression of certain genes can be regulated at the level of transcription. Transcriptional regulation is carried out either negatively (repressors) or positively (activators) by a protein factor.
It is also known that certain specific protein factors regulate translation of specific mRNAs. As indicated hereinabove, it has become evident that RNAs are involved in regulating the expression of specific genes and it has been reported that a small mRNA transcript of 174 bases is produced, upon growing Escherichia coli in a medium of high osmolarity, which inhibits the expression of a gene coding for a protein called Omp F. See Mezuno, et al "Regulation of Gene Expression by a Small RNA Transcript (micRNA) in Escherichia coli: K-12", Proc. Jap. Acad., 59, 335-338 (1983). The inhibition of OmpF protein production by the small mRNA transcript (mic-RNA, i.e. mRNA interfering complementary RNA) is likely due to the formation of a hybrid between the micRNA and the ompF mRNA over a region of approximately 80 bases including the Shine-Dalgarno sequence and the initiation codon.
A similar regulation by a small complementary mRNA has also been described for the Tn10 transposase gene, see Simons et al. "Translational Control of IS10 Transposition", Cell, 34, 683-691 (1983). In this case, however, the gene for the transposase Protein and the gene for the micRNA are transcribed in opposite directions off the same segment of DNA such that the 5'-ends of the transcripts can form a complementary hybrid. The hybrid is thought to inhibit translation of the transposase mRNA. However, the transposase situation is in contrast to the ompF situation in which the ompF gene and the micRNA gene (micF) are completely unlinked and map at 21 and 47 minutes, respectively, on the E. coli chromosomes.
It is an object of this invention to provide a technique useful for the regulation of gene expression of a cell and/or an organism.
It is another object of this invention to provide transformed cells and/or organisms having special properties with respect to the gene expression of the genetic material making up said organisms.
It is yet another object of this invention to provide DNA and viral or plasmid vectors containing the DNA, wherein said DNA is transcribed to produce mRNA which is complementary to and capable of binding or hybridizing to the mRNA produced by said gene to be regulated.
It is a further object of this invention to provide an improved technique and materials useful in connection therewith for the regulation or inhibition of gene expression.
It is also an object of this invention to provide transformed organisms, having been transformed with plasmids or viral vectors containing a gene that produces a micRNA which regulates and/or inhibits the gene expression of a gene located within the host organism.
Another object of this invention is to provide DNA, or vectors including plasmids and viral vectors containing said DNA which is transcribed to produce an mRNA (micRNA) which is complementary to and capable of binding or hybridizing with the mRNA transcribed by the gene to be regulated.